ABSTRACTThe initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the presence of ectopic origin copies may interfere with proper genome duplication. Bacteriophage λ was used as a model system. To this end, the outcome of an infection of an E. coli strain harboring ectopic copies of the λ origin region was analyzed. By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication. These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation.

genes-06-00436-f001: Effect of harboring ectopic copies of λOri on the growth profile of HBT after λ infection. The turbidity at OD600nm of HBT, an E. coli K12 thy− derivative of HB101(recA13, leu, pro), and that of its derivative HBT(pOri1) harboring the plasmid pOri1, were determined at indicated times following λ infection. The growth profile of an uninfected HBT culture was also determined and is shown as a control.

Mentions:
It has been well established that initiation of DNA replication in bacteriophage λ depends on the presence of a single viral origin of DNA replication (λOri), which serves as a landing pad for the formation of a DNA-protein complex that facilitates the recruitment of a helicase around the λOri DNA region [2,7]. λOri is located within the DNA sequence encoding the O protein, an important player in the initiation of λ DNA replication. The minimum λOri region is contained within a 164 bp sequence that consists of two parts: Four 19 bp repeating units, (iterons I–IV, separated from one another by short spacers), on the left; and a 49 bp region on the right (next to iteron I), which is A-T rich and highly strand asymmetric [19]. Both parts of the λOri region are required for replication initiation as demonstrated by the behavior of Ori− mutations in both segments [20,21]. To test if an ectopic λOri, contained in a pBR322-derived recombinant plasmid named pOri1 [10], could interfere with this interaction during a normal λ infection, we first compared the growth profiles of isogenic bacterial E. coli strains containing or not pOri1, following λ infection. The results shown in Figure 1 indicate that upon λ infection, while the growth profile of the strain lacking pOri1 was typical of a lytic infection, the strain containing pOri1 grew as if had not been infected at all by the virus. In fact, its growth profile was similar to that of an uninfected culture. These results suggested that the presence of pOri1 somehow protected the bacteria from a lytic infection. To ascertain that this resistance was dependent solely on the presence of the λOri region, bacteria containing only the parental plasmid were infected under the same conditions as above. As expected, the resulting growth profile was indistinguishable from a normal lytic infection (Figure S1). We also investigated the growth profile of an isogenic bacterial strain harboring two ectopic copies of the λOri region (pOri2). We did not notice any significant difference between the strains harboring one or two copies of the λOri region (Figure S2). This result suggested that one ectopic copy of the λOri region per plasmid is sufficient to provide resistance to λ infection.

genes-06-00436-f001: Effect of harboring ectopic copies of λOri on the growth profile of HBT after λ infection. The turbidity at OD600nm of HBT, an E. coli K12 thy− derivative of HB101(recA13, leu, pro), and that of its derivative HBT(pOri1) harboring the plasmid pOri1, were determined at indicated times following λ infection. The growth profile of an uninfected HBT culture was also determined and is shown as a control.

Mentions:
It has been well established that initiation of DNA replication in bacteriophage λ depends on the presence of a single viral origin of DNA replication (λOri), which serves as a landing pad for the formation of a DNA-protein complex that facilitates the recruitment of a helicase around the λOri DNA region [2,7]. λOri is located within the DNA sequence encoding the O protein, an important player in the initiation of λ DNA replication. The minimum λOri region is contained within a 164 bp sequence that consists of two parts: Four 19 bp repeating units, (iterons I–IV, separated from one another by short spacers), on the left; and a 49 bp region on the right (next to iteron I), which is A-T rich and highly strand asymmetric [19]. Both parts of the λOri region are required for replication initiation as demonstrated by the behavior of Ori− mutations in both segments [20,21]. To test if an ectopic λOri, contained in a pBR322-derived recombinant plasmid named pOri1 [10], could interfere with this interaction during a normal λ infection, we first compared the growth profiles of isogenic bacterial E. coli strains containing or not pOri1, following λ infection. The results shown in Figure 1 indicate that upon λ infection, while the growth profile of the strain lacking pOri1 was typical of a lytic infection, the strain containing pOri1 grew as if had not been infected at all by the virus. In fact, its growth profile was similar to that of an uninfected culture. These results suggested that the presence of pOri1 somehow protected the bacteria from a lytic infection. To ascertain that this resistance was dependent solely on the presence of the λOri region, bacteria containing only the parental plasmid were infected under the same conditions as above. As expected, the resulting growth profile was indistinguishable from a normal lytic infection (Figure S1). We also investigated the growth profile of an isogenic bacterial strain harboring two ectopic copies of the λOri region (pOri2). We did not notice any significant difference between the strains harboring one or two copies of the λOri region (Figure S2). This result suggested that one ectopic copy of the λOri region per plasmid is sufficient to provide resistance to λ infection.

Bottom Line:
Bacteriophage λ was used as a model system.By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication.These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation.

ABSTRACTThe initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the presence of ectopic origin copies may interfere with proper genome duplication. Bacteriophage λ was used as a model system. To this end, the outcome of an infection of an E. coli strain harboring ectopic copies of the λ origin region was analyzed. By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication. These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation.